This invention relates to soybean protein products having significantly lower stachyose content as a function of using the seeds of a soybean line having a heritable seed stachyose content of less than 50 xcexcmol/g (dry seed basis). The present invention also relates to such low stachyose-containing soybeans.
Raffinose saccharides are a group of D-galactose-containing oligosaccharides of sucrose that are widely distributed in plants. Raffinose saccharides are characterized by having the general formula: [0-xcex2-D-galactopyranosyl-(1xe2x86x926)n-xcex1-glucopyranosyl-(1xe2x86x922)-xcex2-D-fructofuranoside where n=0 through n=4 are known respectively as sucrose, raffinose, stachyose, verbascose, and ajugose.
Extensive botanical surveys of the occurrence of raffinose saccharides have been reported in the scientific literature [see Dey, P. M. In Biochemistry of Storage Carbohydrates in Green Plants, Academic Press, London, (1985) pp 53-129]. Raffinose saccharides are thought to be second only to sucrose among the nonstructural carbohydrates with respect to abundance in the plant kingdom. In fact, raffinose saccharides may be ubiquitous, at least among higher plants. Raffinose saccharides accumulate in significant quantities in the edible portion of many economically significant crop species. Examples include soybean (Glycine max L. Merrill), sugar beet (Beta vulgaris), cotton (Gossypium hirsutum L.), canola (Brassica sp.) and all of the major edible leguminous crops including beans (Phaseolus sp.), chick pea (Cicer arietinum), cowpea (Vigna unguiculata), mung bean (Vigna radiata), peas (Pisum sativum), lentil (Lens culinaris) and lupine (Lupinus sp.).
The biosynthesis of raffinose saccharides has been fairly well characterized [see Dey, P. M. In Biochemistry of Storage Carbohydrates in Green Plants (1985)]. The committed reaction of raffinose saccharide biosynthesis involves the synthesis of galactinol (O-xcex1-D-galactopyranosyl-(1xe2x86x921)-myo-inositol) from UDP galactose and myo-inositol. The enzyme that catalyzes this reaction is galactinol synthase. Synthesis of raffinose and higher homologues in the raffinose saccharide family from sucrose is thought to be catalyzed by distinct galactosyltransferases (e.g., raffinose synthase, stachyose synthase, etc.).
Although abundant in many species, raffinose saccharides are an obstacle to the efficient utilization of some economically important crop species. Raffinose saccharides are not digested directly by animals, primarily because xcex1-galactosidase is not present in the intestinal mucosa [Gitzelmann and Auricchio Pediatrics (1965) 36:231-236, Rutloff et al Nahrung (1967) 11:39-46]. However, microflora in the lower gut are readily able to ferment the raffinose saccharides which results in an acidification of the gut and production of carbon dioxide, methane and hydrogen [Murphy et al. (1972) J. Agr. Food Chem. 20:813-817, Cristofaro et al. In Sugars in Nutrition, (1974) Chapter 20, 313-335, Reddy et al. J. Food Science (1980) 45:1161-1164). The resulting flatulence can severely limit the use of leguminous plants in animal, including human, diets. It is unfortunate that the presence of raffinose saccharides restricts the use of soybeans in animal, including human, diets because otherwise this species is an excellent source of protein and fiber.
The soybean is well-adapted to machinery and facilities for harvesting, storing and processing that are widely available in many parts of the world. In the U.S. alone, approximately 28 million metric tons of meal were produced in 1988 (Oil Crops Situation and Outlook Report, April 1989, U.S. Dept of Agriculture, Economic Research Service). Typically, hulls are removed and then the oil is extracted with hexane in one of several extraction systems. The remaining defatted flakes can then be used for a variety of commercial soy protein products [see Soy Protein Products, Characteristics, Nutritional Aspects and Utilization (1987) Soy Protein Council]. Foremost among these in volume of use is soybean meal, the principle source of protein in diets used for animal feed, especially those for monogastric animals such as poultry and swine.
Although the soybean is an excellent source of vegetable protein, there are inefficiencies associated with its use that appear to be due to the presence of raffinose saccharides. Compared to maize, the other primary ingredient in animal diets, gross energy utilization for soybean meal is low [see Potter and Potchanakorn In Proceedings World Soybean Conference III, (1984) 218-224]. For example, although soybean meal contains approximately 6% more gross energy than ground yellow corn, it has about 40 to 50% less metabolizable energy when fed to chickens. This inefficiency of gross energy utilization does not appear to be due to problems in digestion of the protein fraction of the meal, but rather due to the poor digestion of the carbohydrate portion of the meal. It has been reported that removal of raffinose saccharides from soybean meal by ethanol extraction results in a large increase in the metabolizable energy for broilers [Coon, C. N. et al. Proceedings Soybean Utilization Alternatives, University of Minnesota, (1988) 203-211]. Removal of the raffinose saccharides was associated with increased utilization of the cellulosic and hemicellulosic fractions of the soybean meal.
A variety of processed vegetable protein products are produced from soybean. These range from minimally processed, defatted items such as soybean meal, grits, and flours to more highly processed items such as soy protein concentrates and soy protein isolates. In other soy protein products the oil is not extracted, full-fat soy flour for example. In addition to these processed products, there are also a number of speciality products based on traditional Oriental processes, which utilize the entire bean as the starting material. Examples include soy milk, soy sauce, tofu, natto, miso, tempeh, and vuba.
Examples of use of soy protein products in human foods include soy protein concentrates, soy protein isolates, textured soy protein, soy milk, and infant formula. Facilities and methods to produce protein concentrates and isolates from soybeans are available across the world. One of the problems faced by producers of soy protein concentrates and isolates is the challenge of selectively purifying the protein away from the raffinose saccharides. Considerable equipment and operating costs are incurred as a result of removing the large amounts of raffinose saccharides that are present in soybeans.
The problems and costs associated with raffinose saccharides could be reduced or eliminated through the availability of genes that confer a reduction of raffinose saccharide content of soybean seeds. Such genes could be used to develop soybean varieties having inherently reduced raffinose saccharide content. Soybean varieties with inherently reduced raffinose saccharide content would improve the nutritional quality of derived soy protein products and reduce processing costs associated with the removal of raffinose saccharides. Said low raffinose saccharide soybean varieties would be more valuable than conventional varieties for animal and human diets and would allow mankind to more fully utilize the desirable nutritional qualities of this edible legume.
Efforts have been made to identify soybean germplasm that may contain genes that confer a low seed raffinose saccharide content phenotype. Surveys of the soybean germplasm collection, including Glycine max, Glycine soja, and Glycine hirsutum, tentatively identified PI lines that seemed to offer the potential for reducing raffinose saccharide content via conventional breeding [see Hymowitz, T., et al. Comm. In Soil Science and Plant Analysis (1972) 3:367-373, Hymowitz, T., et al. Agronomy J. (1972) 64:613-616, Hymowitz, T., and Collins, F. I. Agronomy J. (1974) 66:239-240, Openshaw, S. J., and Hadley, H. H. Crop Science (1978) 18:581-584, Openshaw, S. J., and Hadley, H. H. Crop Science (1981) 21:805-808, and Saravitz (1986) Ph.D. Thesis, North Carolina State University, Horticultural Science Department]. However, when assayed under identical analytical conditions, none of the lines suggested in these prior surveys proved to be significantly lower in raffinose saccharide content than the currently available elite soybean lines. The primary reason for this may be due to the instability of the low raffinose saccharide phenotype. Results from germplasm collection surveys are highly influenced by the quality of the seed obtained from the collection. This is particularly true for raffinose saccharides in that seed carbohydrate composition has been shown to be influenced by seasonal, genetic and environmental factors [Jacorzynski, B. and Barylko-Pikielna, N. Acta Agrobotanica (1983) 36:41-48, Saravitz (1986) Ph.D. Thesis, North Carolina State University, Horticultural Science Department]. Furthermore, seed storage conditions prior to analysis can also influence the composition [Ovacharov and Koshelev Fiziol. Rast. (1974) 21:969-974, Caffrey et al. Plant Physiol. (1988) 86:754-758, Schleppi and Bums Iowa Seed Science (1989) 11:9-12]. As a result, the potential exists for falsely identifying soybean germplasm whose reduced raffinose saccharide content is not heritable, but rather dur to the environment in which the seeds were produced or stored prior to analysis. Collectively, these factors have severely limited efforts to identify soybean genes that reduce raffinose saccharide content.
The difficulty and unreliability of screens for raffinose saccharide content is reflected by the paucity of publicly available soybean carbohydrate data as compared to protein and oil quality data. For example, the USDA has numerous publications revealing the protein and oil quality contents for almost all (ca. 14,000) of the soybean PI lines in the USDA collection. However, although raffinose saccharide content is known to be a serious problem in soybeans, very little of the PI collection has actually been screened for this trait.
Demonstration of the stability of a low raffinose saccharide phenotype in subsequent generations (heritability of the phenotype) is required if the germplasm is to be of any utility in improving seed quality. It is therefore essential that any putative germplasm source be regrown to obtain fresh seed and reassayed (with appropriate lines as experimental controls) before it is declared as a potential source of low raffinose saccharide genes. Once the heritability (stability) of the phenotype is demonstrated, it is desirable to determine the inheritance (number and nature of genes that are involved) of the low raffinose saccharide phenotype. Heritability and inheritance information is extremely valuable for attempts to breed new soybean varieties that contain the low raffinose saccharide trait.
In light of the above described factors, it is apparent that soybean plants with heritable, substantially reduced raffinose saccharide content useful for preparing soy protein products with an improved carbohydrate content are needed. Heretofore, the only means to acheive a desirable raffinose saccharide content was to physically and/or chemically treat the soybean.
The present invention comprises soybean line(s) with a heritable phenotype of a seed stachyose content of less than 50 xcexcmol/g or a seed total raffinose saccharide content of less than 120 xcexcmol/g. Soybean seeds with this stachyose content are also an embodiment of this invention. Soybean line(s) having a genotype at the Stc1 locus that confers a phenotype of a seed stachyose content of less than 50 xcexcmol/g or a total raffinose saccharide content of less than 120 xcexcmol/g is also an embodiment of this invention. Preferred are seeds, plant lines producing seeds, plants producing seeds and the progeny of such plant lines, plants and seeds that have a heritable phenotype of a seed stachyose content of less than 30 xcexcmol/g or less than 15 xcexcmol/g, respectively. A further embodiment of the invention is a soybean line(s) or seeds having the stachyose or total raffinose saccharide contents set forth above and a seed protein content of greater than 42%.
A further embodiment of the invention is a method of using a soybean line having a heritable phenotype of a seed stachyose content of less than 50 xcexcmol/g, the method comprising processing said seeds to obtain a desired soy protein product. A further embodiment of the invention is a method of making a soy protein product comprising processing seeds of a soybean line having a heritable phenotype of a seed stachyose content of less than 50 xcexcmol/g. Preferred embodiments are methods of using a soybean line having a genotype at the Stc1 locus phenotype of a seed stachyose content of less than 50 xcexcmol/g, 30 xcexcmol/g, and 15 xcexcmol/g, respectively to process said seeds to obtain a desired soy protein product. Additional preferred embodiments are methods of making a soy protein product comprising processing seed of a soybean line having a genotype at the Stc1 locus phenotype that confers a seed stachyose content of less than 50 xcexcmol/g, less than 30 xcexcmol/g, or less than 15 xcexcmol/g respectively.
The present invention further comprises methods for making a full fat soy protein product, the method comprising: (a) cracking seeds from a soybean line having a heritable phenotype of a seed stachyose content of less than 50 xcexcmol/g to remove the meats from the hulls; (b) flaking the meats obtained in step a to obtain a desired flake thickness; (c) heat-denaturing the flakes obtained in step (b) to obtain a desired Nitrogen Solubility Index; and (d) grinding the denatured flakes of step (c) to obtain a desired particle size. The present invention further comprises adding soybean hulls to the product of step (c) to obtain a full fat soy protein product having a maximum fibre content of 7% at a moisture content of 12%.
The present invention further comprises a method of making a defatted soy protein product comprising: (a) cracking seeds from a soybean line having a heritable phenotype of a seed stachyose content of less than 50 xcexcmol/g to remove the meats from the hulls; (b) flaking the meats obtained in step (a) to obtain a desired flake thickness; (c) contacting the full flakes obtained in step (b) with a solvent to extract oil from the flakes to a desired content level; (d) heat-denaturing the defatted flakes obtained in step (c) to obtain a desired Nitrogen Solubility Index; and (e) grinding the denatured, defatted flakes obtained in step (d) to obtain a desired particle size. The present invention further comprises adding soybean hulls to the product of step (c) to obtain a defatted soy protein product having a maximum fibre content of 7% at a moisture content of 12%. The heat-denaturing may be accomplished by flash desolventization. Extruding the full fat soy protein product or the defatted soy protein product to texturize or structure the product after the grinding step is also included in the present invention.
The present invention further comprises a method of making a soy protein concentrate product comprising: (a) cracking seeds from a soybean line having a heritable phenotype of a seed stachyose content of less than 50 xcexcmol/g to remove the meats from the hulls; (b) flaking the meats obtained in step (a) to obtain a desired flake thickness ; (c) contacting the full fat flakes obtained in step (b) with a first solvent to extract oil from the flakes to a desired oil content level; (d) contacting the defatted flakes obtained in step (c) with a second solvent to obtain a soy protein concentrate product with a protein content (6.25xc3x97N) of not less than 65% (db). A preferred embodiment of this invention uses an aqueous alcohol solution from 55% to 90% as a second solvent, the soy protein concentrate product obtained in step (d) having a protein content (6.25xc3x97N) of not less than 70% (db). A second preferred embodiment of this invention uses an acidic solution of pH 4 to pH 5 as a second solvent.
The present invention further comprises a method of making an isoelectric soy protein isolate product comprising: (a) cracking seeds from a soybean line having a heritable phenotype of a seed stachyose content of less than 50 xcexcmol/g to remove the meats from the hulls; (b) flaking the meats obtained in step (a) to obtain a desired flake thickness; (c) contacting the full fat flakes obtained in step (b) with a first solvent to extract oil from the flakes to a desired oil content level; (d) contacting the defatted flakes obtained in step (c) with an aqueous solution of pH 8 to pH 9; (e) separating the soluble and insoluble fractions of the product of step (d) by physical means; (f) adjusting the pH of the soluble fraction obtained in step (e) to obtain a protein precipitate; (g) separating the protein precipitates of step (f) from the soluble fraction by physical means to obtain a soy protein isolate; (h) washing the product of step (g) with water; and (i) spray-drying the washed product of step (h) to obtain an isoelectric soy protein isolate product. A further embodiment of this invention comprises mixing the soy protein isolate product obtained in step (i) with sufficient alkali to increase the solubility of the product to a desired level.
The present invention further comprises a method of making a pet food product comprising: (a) combining farinaceous materials, a soy protein product derived from the processing of soybean seed with a heritable stachyose content of less than 50 xcexcmol/g at an inclusion rate of less than 41%, animal fat, vitamins, minerals, and salt into a mixture; (b) extruding the mixture of step (a) through a die at an elevated temperature and pressure; (c) portioning the extruded mixture of step (b) into pieces of a desirable size; and (d) drying the products of step (c) to a desirable moisture content preferably a moisture content of less than 10%.
The present invention further comprises a full fat soy protein product derived from the processing of soybean seed with a heritable stachyose content of less than 50 xcexcmol/g, preferably less than 30 xcexcmol/g, or more preferably less than 15 xcexcmol/g. A further embodiment of the invention also contains a protein content of greater than 42% at each of the stachyose content levels stated.
The present invention further comprises an undenatured, defatted soy protein product derived from the processing of soybean seeds with a heritable seed stachyose content of less than 50 xcexcmol/g, preferably less than 30 xcexcmol/g, or more preferably less than 15 xcexcmol/g.
The invention further includes a heat-processed, defatted, desolventized, and toasted soy protein product derived from the processing of soybean seeds with a heritable seed stachyose content of less than 50 xcexcmol/g, preferably less than 30 xcexcmol/g, or more preferably less than 15 xcexcmol/g.
The present invention further comprises a heat-processed, defatted, desolventized and toasted soy protein product that (a) is derived from the processing of soybean seeds with a heritable seed stachyose content of less than 50 xcexcmol/g, preferably less than 30 xcexcmol/g, or more preferably less than 15 xcexcmol/g and (b) has a true metabolizable energy (TMEN) content of greater than 2850 Kcal/Kg (db).
The present invention further comprises a heat-processed, defatted, desolventized and toasted soy protein product that (a) is derived from the processing of soybean seeds with a heritable seed stachyose content of less than 50 xcexcmol/g, preferably less than 30 xcexcmol/g, or more preferably less than 15 xcexcmol/g and (b) is derived from soybean seeds with a protein content of greater than 42%.
The present invention further comprises a heat-processed, defatted, flash-desolventized soy protein product derived from the processing of soybean seeds with a heritable seed stachyose content of less than 50 xcexcmol/g, preferably less than 30 xcexcmol/g, or more preferably less than 15 xcexcmol/g.
The invention further includes an undenatured, defatted, flash desolventized soy protein product that (a) is derived from the processing of soybean seeds with a heritable seed stachyose content of less than 50 xcexcmol/g, preferably less than 30 xcexcmol/g, or more preferably less than 15 xcexcmol/g amd (b) is derived from soybean seeds with a protein content of greater than 42%.
The present invention further comprises a heat-processed, defatted soy protein product derived from the processing of soybean seeds with a heritable seed stachyose content of less than 50 xcexcmol/g, preferably less than 30 xcexcmol/g, or more preferably less than 15 xcexcmol/g. A preferred embodiment of this invention is a protein product that has a Nitrogen Solubility Index of greater than 60, a more preferred embodiment has a Nitrogen Solubility Index of between 20 and 60, and the most preferred embodiment has a Nitrogen Solubility Index of less than 20.
The present invention further comprises a soy protein concentrate product having a protein content (6.25xc3x97N) of not less than 65% (db) produced by the method comprising: (a) cracking seeds from a soybean line having a heritable phenotype of a seed stachyose content of less than 50 xcexcmol/g to remove the meats from the hulls; (b) flaking the meats obtained in step (a) to obtain a desired flake thickness; (c) contacting the full fat flakes obtained in step (b) with a first solvent to extract oil from the flakes to a desired oil content level; (d) contacting the defatted flakes obtained in step (c) with a second solvent to obtain a soy protein concentrate product with a protein content (6.25xc3x97N) of not less than 65% (db).
The present invention further comprises a pet food product that (a) is derived from the processing of soybean seeds with a heritable seed stachyose content of less than 50 xcexcmol/g, preferably less than 30 xcexcmol/g, or more preferably less than 15 xcexcmol/g and (b) has a soybean protein product inclusion rate of between 25% and 41%.
The present invention further comprises mutant soybean lines that have been deposited under the terms of the Budapest Treaty at ATCC (Americal Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852), and bear the following accession numbers:
The present invention further comprises a soybean line derived from a cross between any soybean line, preferably an agronomically elite line, and LR28, LR484 (ATCC 75325), LR3705, LR4271, or LR33*LR28, the cross yielding a soybean line with a heritable phenotype of less than 50 xcexcmol/g seed stachyose content or a seed total raffinose saccharide content of less than 120 xcexcmol/g. Progeny from any pedigree involving LR28, LR484 (ATCC 75325), LR3705, LR4271, or LR33*LR28 as at least one parent, the plant having a heritable phenotype of less than 50 xcexcmol/g seed stachyose or less than 120 xcexcmol/g total raffinose saccharide, are also embodiments of the present invention. Seeds of these crosses displaying these stachyose and total raffinose saccharide levels are also an embodiment of this invention. This invention preferably has an additional characteristic of protein content greater than 42%.
The present invention further comprises a method for producing a soybean protein product derived from processing soybean seeds having a heritable total raffinose saccharide content of less than 120 xcexcmol/g or a stachyose content of less than 50 xcexcmol/g comprising: (a) crossing any soybean line, preferably an agronomically elite soybean line, with LR28, LR484, LR3705, LR4271, or LR33*LR28 to obtain an F1 hybrid; (b) crossing and/or selfing the F1 hybrid for at least one generation to obtain segregating progeny; (c) identifying the progeny of step (b) with a heritable total raffinose saccharide seed content of less than 120 xcexcmol/g or a seed stachyose content of less than 50 xcexcmol/g; and (d) processing the seed selected in step (c) to obtain the desired soybean protein product.
A further aspect of the invention is a method of using a soybean line having a genotype at the Stc1 locus that confers a heritable seed phenotype of less than 50 xcexcmol/g stachyose or less than 120 xcexcmol/g total raffinose saccharide content to produce progeny lines, the method comprising: (a) crossing a soybean plant containing a stc1x allele with any soybean parent, preferably an agronomically elite soybean parent, which does not contain said allele, to yield a F1 hybrid; (b) crossing and/or selfing the F1 hybrid for at least one generation to obtain segregating progeny; (c) identifying the progeny of step (b) with a heritable seed stachyose content of less than 50 xcexcmol/g or a total raffinose saccharide content of less than 120 xcexcmol/g.